Since NADPH-cytochrome P-450 reductase and two forms of cytochrome P-450 from rabbit liver microsomes have already been obtained in highly purified state in this laboratory, we are now turning our attention to the further characterization and study of the catalytic function of these proteins. Attempts will be made to determine the amino acid sequence of each as well as to provide complete physical chracterization. With the reductase, it is important to determine whether FMN or FAD (or perhaps both) function in electron transfer from NADPH to these cytochromes. We will continue our investigations on the position-specific hydroxylation of various substrates, including benzo(a)pyrene, warfarin, steroids, benzphetamine, and other drugs in the reconstituted enzyme system. In addition, attempts will be made to purify to a greater extent the remaining four forms of rabbit liver microsomal cytochrome P-450 which have so far been solubilized and only partially freed from contaminating proteins.